CN103443614B - Use of signal enhancing compounds in electrochemiluminescence detection - Google Patents

Use of signal enhancing compounds in electrochemiluminescence detection Download PDF

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CN103443614B
CN103443614B CN201180062441.9A CN201180062441A CN103443614B CN 103443614 B CN103443614 B CN 103443614B CN 201180062441 A CN201180062441 A CN 201180062441A CN 103443614 B CN103443614 B CN 103443614B
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reagent composition
ecl
reagent
formula
phosphoamide
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CN103443614A (en
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J.斯托克尔
M.温德福尔
A.芬克
B.豪普特曼
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F Hoffmann La Roche AG
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D499/00—Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
    • C07D499/04—Preparation
    • C07D499/10—Modification of an amino radical directly attached in position 6
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/66—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light electrically excited, e.g. electroluminescence
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements

Abstract

The invention concerns methods for the detection of an analyte in a sample by electrochemiluminescence using new reagent compositions. New reagent compositions, reagent kits for measuring electrochemiluminscence (ECL) and electrochemiluminescence detection methods using the new reagent compositions are disclosed. In particular, the invention relates to the use of novel combinations of compounds which can be used in said measurements to provide improved assay performance.

Description

Signal strengthens the purposes of compound in electrochemiluminescence detects
Technical field
The present invention relates to the method using novel agent composition to be detected the analysis thing in sample by electrochemiluminescence.Present application discloses the electrochemical luminous detection method of the described novel agent composition of the novel agent composition for measuring electrochemiluminescence (ECL), kit and use.Specifically, the present invention relates to the purposes that can be used for described measurement and combine to provide the new compound of the mensuration performance of improvement.
Background technology
Measure method some year known of electrochemiluminescence phenomenon.Described method utilizes special metal complex can realize the ability of excited state by oxidation, from the decline of this excited state to ground state, sends electrochemiluminescence.Summary is see Richter, M.M., Chem.Rev.104 (2004) 3003-3036.
At present, there is a variety of commercially available instrument utilizing electrochemiluminescence (ECL) to carry out analysis to measure.The material (ECL-active substance) sending ECL can be caused to be used as ECL label.The example of ECL label comprises organometallics, and such as three-dipyridine-ruthenium (RuBpy) part, wherein metal is such as VII and group VIII metal, comprises Re, Ru, Ir and Os.In ECL process, the material reacted with ECL label is referred to as ECL coreagent in this application.Conventional coreagent for ECL comprises tertiary amine (such as tripropylamine (TPA)), oxalates and persulfate.Light is produced by ECL label or part; The participation of binding reagents in binding interactions is monitored by measuring the ECL sent from ECL label.As selection, the ECL signal from ECL-reactive compound can indicate chemical environment (see such as, United States Patent (USP) 5,641,623 and 5,643,713, the ECL of the existence or destruction which depict monitoring specificity ECL coreagent measures).For more background knowledges of ECL, ECL label for carrying out ECL mensuration, ECL mensuration and instrument, see United States Patent (USP) 5,093,268; 5,147,806; 5,240,863; 5,308,754; 5,324,457; 5,591,581; 5,597,910; 5,679,519; 5,705,402; 5,731,147; 5,786,141; 5,846,485; 5,866,434; 6,066,448; 6,136,268 and 6,207,369 and EP0441875 and disclosed PCT WO97/36931; WO98/12539; WO99/14599; WO99/32662; WO99/58962; WO99/63347; WO00/03233 and WO98/57154.
Commercially available ECL instrument has shown excellent performance.They are owing to comprising its superior sensitivity, dynamic range, precision and being used widely to reasons such as the tolerances of complex sample matrix.Commercially available instrument uses the design based on flow cell, and this design has lasting reusable flow cell.
For determining that the available samples volume analyzing thing is usually restricted, and increasing different thing of analyzing must be determined from a sample.Also require that exploitation is for measuring the laboratory equipment faster of robotization and the more sensitive method for detect analytes.This causes needs highly sensitive with specificity electrochemiluminescence mensuration with for carrying out their method.In addition, also should consider and thing or the relevant improvement of environmental problem of endangering safety.
But the even sensitiveer detection analyzing thing is very favorable.Therefore, target of the present invention improves described known method and reagent composition, and it is specifically surveyed for the analysis quality testing strengthening ECL signal and improvement and electrochemiluminescence operative combination and improves.It is desirable that, the electrochemiluminescence finding to have improvement measures novel signal Contrast agent and/or the compound of performance.
Summary of the invention
The present invention relates to the method being detected the analysis thing measured in sample by electrochemiluminescence in one embodiment, comprise the following steps: hatch together with detection reagent a) sample and electricity consumption chemiluminescent groups marked, b) make to be combined with analyze thing through mark detection reagent be not separated containing the detection reagent through marking analyzing thing, c) the isolated detection reagent through mark is hatched together with reagent composition, described reagent composition comprises: i) at least one coreagent, and ii) be selected from least one compound of the phosphoamide (carbonic acid amide) of formula I and formula II,
Formula I
In formula I, R 1=CH 3, CH 2f, CH 2cl, CH 2cH 3, CHClCH 3, CH 2cH 2cl, C (CH 3) 2cH 3, CH 2cH 2cH 3, CClHCH 2cH 3or CH 2cH 2cH 2cH 3, R 2=H, and R 3=H,
Formula II
D) galvanochemistry triggers luminous release, and e) determines electrochemiluminescence (ECL) signal, analyte thus.
The invention still further relates to the reagent composition for determining ECL, it comprises the compound of phosphoamide i) being selected from formula I and formula II, and ii) at least one coreagent.
The invention still further relates to reagent mixture, it comprises the reagent composition for determining ECL, it comprises the compound of phosphoamide i) being selected from formula I and formula II, and ii) at least one coreagent, sample to be studied and at least one electricity consumption chemiluminescent groups mark detection reagent.
The invention still further relates to the kit for measuring ECL, it comprises the reagent composition for determining ECL, and described reagent composition comprises compound and the ii of phosphoamide i) being selected from formula I and formula II) at least one coreagent.
According to below to the specific descriptions of some preferred embodiment, will more completely understand the present invention and its other target, feature and advantage.
Embodiment
Unless otherwise noted, enforcement of the present invention can adopt the molecular biology in art technology (comprising recombinant technology), microbiology, cell biology, biological chemistry and immunologic routine techniques.This kind of technology is fully explained in the following documents, such as " Molecular Cloning:A LaboratoryManual ", the second edition (Sambrook etc., 1989); " Oligonucleotide Synthesis " (M.J.Gait compiles, 1984); " Animal Cell Culture " (R.l.Freshney compiles, 1987); " Methods inEnzymology " (Academic Press, Inc.); " Current Protocols in Molecular Biology " (volume such as F.M.Ausubel, 1987 and regular update); " PCR:The Polymerase Chain Reaction ", (volume such as Mullis, 1994).
Unless otherwise defined, the technology used in the application and scientific terminology have identical implication with the usual understanding of those skilled in the art.The Dictionary ofMicrobiology and Molecular Biology of the people such as Singleton, the 2nd edition, J.Wiley & Sons, New York (1994); March, Advanced Organic Chemistry Reactions, Mechanisms andStructure, the 4th edition, John Wiley & Sons, New York (1992); Lewin, B., Genes V, publishes (1994) by Oxford University Press, ISBN0-19-8542879); The people such as Kendrew, J. (volume), The Encyclopedia of Molecular Biology, published by Blackwell ScienceLtd. (1994), ISBN0-632-02182-9); And Meyers, R.A. (ed.), Molecular Biology andBiotechnology:a Comprehensive Desk Reference, by VCH Publishers, Inc. (1995) are published, ISBN1-56081-5698) provide to those skilled in the art and the generality of the many terms used in the application is instructed.
Its full content is incorporated to the application by all lists of references (comprising patented claim and publication) by way of reference that quote in the application.
definition
As used in this application, following every term has the implication relevant with it in this section.
Article " one " and " one " refer in this application one or more than one (i.e. at least one) article grammar object.For example, " analyze thing for one " and mean that one is analyzed thing or analyzes thing more than one.Term " at least " is used to indicate optionally, can there is other object one or more.For example, optionally, the array comprising at least two zone of dispersions can comprise two or more discrete test sections.
Statement " one or more " refers to 1-50, preferred 1-20, also preferably 2,3,4,5,6,7,8,9,10,12 or 15.
The example of " phosphoamide " and chemical constitution thereof is listed in the table below in 1.
Table 1: phosphoamide has following common structure, unless otherwise noted:
The application formula I used represents:
R in formula I 1=CH 3, CH 2f, CH 2cl, CH 2cH 3, CHClCH 3, CH 2cH 2cl, C (CH 3) 2cH 3, CH 2cH 2cH 3, CClHCH 2cH 3or CH 2cH 2cH 2cH 3, R 2=H, and R 3=H.
The application formula II used represents following structure, is called 2-Pyrrolidone, as shown in numbering in table 1 24.
Embodiment of the present invention can be used for testing the various sample that may comprise interested analysis thing or activity.Described sample can be solid, emulsion, suspension, liquid or gas form.They can be, but be not limited to comprise the sample of following material or come from the sample of human or animal: such as, cell (survival or dead) and cell-derived product, immortalized cells, cell fragment, cell fraction (cell fraction), lysate, organelle, cell membrane, hybridoma, cell culture supernatant (comprising from the supernatant of antibody tormation biosome as hybridoma), waste water or potable water, food, beverage, pharmaceutical composition, blood, serum, blood plasma, hair, sweat, urine, excreta, ight soil, saliva, tissue, biopsy, effluent, the sample of separation and/or classification, the liquid of separation and/or classification, organ, plant, plant part, plant by-product, soil, water, supply water, water source, from the residue that fluid (gas and liquid) leaches, the swipes of taste flat, absorbent material, gel, cytoskeleton, unassorted sample, unassorted lysate, nucleus, core fraction, chemicals, chemical solution, structure-biological component, bone (ligament, tendon) component, the bone component of separation and/or classification, hair component and/or separator, skin, skin samples, skin components, corium, endodermis, eukaryotic, prokaryotic, fungi, yeast, immunocyte, medicine, medicine, oil, extract, mucus, sewage, environmental sample, organic solvent or air.In one embodiment, sample may further include, such as, and water, alcohol, acetonitrile, dimethyl sulfoxide (DMSO), dimethyl formamide, N-methyl-pyrrolidon, methyl alcohol or other organic solvents.
The application's " sample " used obtains for the object of in-vitro evaluation.As those of skill in the art can understand, any described evaluation is carried out all in vitro.If sample is Patient Sample A, then then discard Patient Sample A.The material transfer of Patient Sample A only for in-vitro diagnosis method of the present invention, and is not got back in patient body by Patient Sample A.
Can include but not limited to by tested quantitative analysis thing, full cell, cell surface antigen, protein complex, cell signal factor and/or assembly, second messenger, second messenger's signal factor and/or assembly, sub-cellular particles (such as organelle or membrane-bound fragment), virus, prion, dust mite or its fragment, viroid, immune factor, antibody, antibody fragment, antigen, haptens, fatty acid, nucleic acid (and synthetic analogues), ribosomes, protein (with the analog of synthesis), lipoprotein, polysaccharide, inhibitor, accessory factor, haptens, cell receptor, receptors ligand, lipopolysaccharides, glycoprotein, peptide, polypeptide, enzyme, zymolyte, enzyme product, nucleic acid processive enzyme (such as polymerase, nuclease, integrase, ligase, unwindase, telomerase etc.), protein processive enzyme (such as proteinase, kinases, protein phosphatase, general peptide-protein ligase etc.), products of cellular metabolism, endocrine factor, paracrine factor, autocrine factor, cell factor, hormone, medicament, medicine, medicine, synthetic organic molecule, organic-metal molecules, sedative, barbiturate, alkaloid, steride, vitamin, amino acid, sugar, agglutinin, recombinant or derived protein, biotin, avidin, the inorganic molecule existed in streptavidin or sample.
Full cell can be animal, plant or bacterium, and can be alive or dead.Example comprises phytopathogen such as fungi and nematode.Term " sub-cellular particles " is intended to comprise, such as, and subcellular organelle, membrane granule, cell wall fragments, ribosomes, the multienzyme complex of Tathagata self destruction cell, and other particle that can be derived from biosome alive.Nucleic acid comprises, such as, and chromosomal DNA, plasmid DNA, viral DNA, and the recombinant DNA being derived from multiple source.Nucleic acid also comprises RNA, such as mRNA, rRNA and transfer RNA.Polypeptide comprises such as, enzyme, transport protein, receptor protein, and structural proteins are as virus capsid protein.Preferred polypeptide is enzyme and antibody.Particularly preferred polypeptide is monoclonal antibody.Hormone comprises such as, insulin and T4 thyroid hormone.Medicine comprises such as, cardiac glycoside.Certainly, be included in chemically similar to biological substance synthetic within the scope of the invention, as improvement on synthesis, nucleic acid, and synthesis film, vesica and liposome.Aforementionedly be not intended to the comprehensive list being applicable to biological substance of the present invention, and be only intended to example broad range of the present invention is described.
Also common, interested analysis thing is with 10 -3following volumetric molar concentration exists, and such as at least lowly reaches 10 -18volumetric molar concentration.
(ASR) it must be understood that as the molecule or the biomolecule (such as protein or antibody) that have with analyze thing specific binding capacity " to analyze thing specific reagent " according to term of the present invention.(ASR) is can for the identification of a class biomolecule of the amount with the independent chemical substance measured in biological specimen " to analyze thing specific reagent ".ASR is such as antibody (polyclone or monoclonal antibody), specific receptor albumen, part, nucleotide sequence and similar reagents, described similar reagents be intended to by carry out specific binding or chemical reaction with the material in sample and in diagnostic application to identify and chemical substance independent in quantitative biological specimen or part.In brief, analyzing thing specific reagent is the active component measured.ASR will meet the affinity standard and specific criteria that are combined with analysis thing.
Term " antibody " uses with most broad sense, and clearly contains monoclonal antibody (comprising full length monoclonal antibodies), polyclonal antibody, multi-specificity antibody (such as bispecific antibody) and antibody fragment.Various forms of antibody structure contained in term " antibody ", comprises complete antibody and antibody fragment.Antibody of the present invention is people's antibody in one embodiment, humanized antibody, chimeric antibody, be derived from other animal as the antibody of mouse, goat or sheep, monoclonal or polyclonal antibody, or removes the antibody of T cell antigen.The genetic engineering of antibody is such as described in Morrison, S.L., et al., Proc.Natl.Acad Sci.USA81 (1984) 6851-6855; US5,202,238 and US5,204,244; Riechmann, L., et al., Nature332 (1988) 323-327; Neuberger, M.S., et al., Nature314 (1985) 268-270; In Lonberg, N., Nat.Biotechnol.23 (2005) 1117-1125.
Any antibody fragment keeping the above-mentioned standard analyzing thing specific reagent can be used.Antibody is produced by the operation of this area, such as, as Tijssen (Tijssen, P., Practice and theory of enzymeimmunoassays, 11, Elsevier Science Publishers B.V., Amsterdam, whole book, especially 43-78 page) described in operation.In addition, those of skill in the art know the method based on immuno absorbence that can be used for specific isolation antibody.By these means, the quality of antibody and then its performance in immunoassays can be improved (Tijssen, P., the same, 108-115 page).
" detection reagent " according to the present invention comprises the analysis thing specific reagent (ASR) of electricity consumption chemiluminescent groups mark, or the analysis thing homologue of electricity consumption chemiluminescent groups mark.According to test form, those of skill in the art are known must select which kind of to detect reagent for different mensuration forms (such as sandwich style mensuration, dual antigen bridge joint measure (DAGS), competition assay, homogeneous determination, heterogeneous assays).Detection reagent in heterogeneous immunoassays can be such as antibody.Well known by persons skilled in the art is that detection reagent can immobilization in solid phase.In one embodiment, the method detecting by electrochemiluminescence the analysis thing measured in sample can be carried out with homogeneous determination.In one embodiment, the method can be carried out with heterogeneous assays.In one embodiment, the method can measure form with sandwich style and carries out.In one embodiment, the method can be carried out with competition assay form.Also in one embodiment, the method can be carried out with dual antigen bridge joint mensuration (DAGS) form.Known immunoassay format specifically describes in book Price, C.P.and Newman, D.J., Principles and Practiceof Immunoassay, in 2nd ed. (1997).
The application's term used " label " refers to any material that can produce detectable signal (be no matter visibly or by using the instrument be applicable to).Be applicable to various label of the present invention and include but not limited to the compound of chromogen, fluorescence, chemiluminescent or electrochemiluminescence, catalyzer, enzyme, zymolyte, dyestuff, colloidal metal and non-metallic particle and organic polymer latex particle.
Term " luminescence " refers to any light transmitting not deriving energy from the temperature of the energy (such as, electromagnetic radiation source, chemical reaction, mechanical energy).Usually, this source causes the electronics of atom to move to " exciting " higher-energy state from lower energy state; Then electronics falls after rise to discharging this energy compared with during low-energy state with radiative form at it.This kind of light transmitting occurs usually in the visible or near visible range of electromagnetic spectrum.Term " luminescence " includes but not limited to this kind of smooth emission phenomena such as phosphorescence, fluorescence, bioluminescence, radioluminescence, electroluminescence, electrochemiluminescence and thermoluminescence.
Term " luminous marker " refers to the label generating luminous signal, and described luminous signal does not such as derive the light transmitting of energy from the temperature of emissive source.Luminous marker can be such as fluorescence molecule, phosphorescent molecules, radioluminescence molecule, luminous sequestrant, phosphorus or phosphorus-containing compound or quantum dot.
" electrochemiluminescence mensuration " or " ECLA " are the electrochemical gagings that a kind of label by being connected with detection reagent (target molecule) detects the analyte molecule of combination.Electrode electro Chemical starts and the luminescence detecting the chemical markers that reagent is connected.By the light that photo-detector measurement is launched by label, and the existence of the analyte molecule/target complexes of its instruction combination or amount.ECLA method is recorded in such as United States Patent (USP) 5,543,112; 5,935,779; With 6,316, in 607.Can, in order to accurate and sensitive measurement, for different analyte molecule concentration, signals-modulating be maximized.
In ECLA operation, particulate can be suspended in the sample to which with effective bound analyte.Such as, particle can have the diameter of 0.05 μm-200 μm, 0.1 μm-100 μm or 0.5 μm-10 μm, and can the surface component of analyte binding molecule.A kind of frequent use ECLA system ( rocheDagnsotics, Germany) in, this particulate has the diameter of about 3 μm.Particulate can be formed by following material: the oxide of crosslinked starch, glucosan, cellulose, protein, organic polymer, styrol copolymer such as styrene/butadiene copolymers, acrylonitrile/butadiene/styrene multipolymer, vinylacetyl acrylate copolymer, vinyl chloride/acrylate copolymer, inert inorganic particle, chromium dioxide, iron, silicon dioxide, silica mixture, proteinaceous material or its potpourri, include but not limited to sepharose 4B, latex beads, shell core particle etc.Preferably, particulate is monodispersed, and can be magnetic, such as paramagnetic bead.See such as United States Patent (USP) 4,628,037; 4,965,392; 4,695,393; 4,698,302; With 4,554,088.Can with scope for about 1-10, the amount of 000 μ g/ml, preferably 5-1,000 μ g/ml uses particulate.
Statement " interested " refers to the analysis thing that may associate that analyze or determine or material.
" detection " comprises any detection means, comprises directly and indirect detection.Term " detection " uses with most broad sense, comprises both the quantitative and qualitative analysis analyzing thing measures, analyte in the application.In one aspect, use if the detection method described in the application is to identify a small amount of existence of interested analysis thing in sample.On the other hand, described method can be used to quantize to analyze in sample the amount of thing.
" reduction " or " suppression " is compared with object of reference, reduces or reduces activity, function and/or amount.Reduce or suppress to mean to cause preferably 10% or larger, more preferably 25% or larger, and most preferably 50%, 75%, 90%, 95% or larger the ability of overall reduction.
" enhancing " such as " enhancing specific signals " or " strengthening ECL signal " improves compared with object of reference or promote activity, function and/or amount.Improve or promote refer to cause overall to improve preferably more than 10%, the ability of more preferably more than 25%, most preferably more than 50%.
Term " is determined " in this application for quantitative and qualitative analysis detect analytes, and can comprise the amount and/or concentration determining to analyze thing.
Term " measurement " is assessment or quantification (such as length or the quality) process relative to the magnitude of measuring unit's (such as rice or kilogram) in science.Measure and use reference point, other things can be assessed for this reference point.The specific outcome (determined value) that also can be used for referring to obtain from measuring process measured in term.It is the basis of comparing.Those of skill in the art know the materials and methods for making measuring-signal or determined value associate with concentration.
The reagent supporting that ECL-signal produces is comprised, such as coreagent, the buffering agent controlled for pH and other optional component according to " reagent composition " of the present invention or " ECL-reagent composition ".Those of skill in the art know the component be present in reagent composition needed for the generation of ECL signal in electrochemical luminous detection method.
The application's " aqueous solution " used is particle, material or liquid compound homogeneous phase solution soluble in water.Aqueous solution also can comprise organic solvent.Organic solvent is well known by persons skilled in the art, such as methyl alcohol, ethanol or dimethyl sulfoxide (DMSO).As used in this application, will also be understood that aqueous solution can comprise the organic solvent of 50% at the most.
The material participating in ECL mark in ECL process is referred to as ECL " coreagent " in this application.Conventional coreagent for ECL comprises tertiary amine (such as tripropylamine (TPA)), oxalates and persulfate.Those of skill in the art know the available coreagent had for electrochemical luminous detection method.
" solid phase ", also referred to as " solid support ", insoluble, functionalized polymeric material, can to its attachment or covalent bond (often via connection base) library constructs or reagent with immobilization or allow that they easily separate (by filtering, centrifugal, cleaning etc. carry out) by excessive reagent, soluble reaction accessory substance or solvent.Solid phase for the inventive method is extensively recorded in (see such as Butler, J.E., Methods22 (2000) 4-23) in prior art.Term " solid phase " refers to non-fluid substance, and comprises the particle (comprising particulate and pearl) be made up of material such as polymkeric substance, metal (paramagnetism, ferromagnetic particle), glass and pottery; Gelatinous mass is silica gel, alumina gel and polymer gel such as; The kapillary can be made up of polymkeric substance, metal, glass and/or pottery; Zeolite and other porous mass; Electrode; Microtiter plate; Solid bar; And little Chi, pipe, chip or other spectrometer sampling receptacle.The solid phase components measured is with the difference measuring the inert solid surface that can contact, and " solid phase ", in its surface containing at least one module, this module is intended to and catches antibody or catch interaction of molecules.Solid phase can be fixing component, such as pipe, bar, little Chi, chip or microtiter plate, or can be unfixed component, such as pearl and particulate.Particulate also can as the solid phase for homogeneous determination form.The multiple particulate of allowing protein and the non-covalent or covalent attachment of other material can be used.This kind of particle comprises polymer beads such as polystyrene and poly-(methyl methacrylate); Gold grain such as gold nano grain and gold colloid; And ceramic particle such as tripoli, glass and metal oxide particle.See such as Martin, C.R. etc., AnalyticalChemistry-News & Features70 (1998) 322A-327A, it is incorporated to the application by way of reference.
Term " chip ", " biochip ", " polymer chip " or " protein-chip " commutative use, and refer to that described substrate can be a part for silicon chip, nylon strip, plastic strip or glass slide in the set of the substrate (such as solid phase) the shared upper a large amount of probes, mark or the biochemical markers that arrange.
method:
In one embodiment, the present invention relates to the method being detected the analysis thing measured in sample by electrochemiluminescence, comprise the following steps: hatch together with detection reagent a) sample and electricity consumption chemiluminescent groups marked, b) make to be combined with analyze thing through mark detection reagent be not separated containing the detection reagent through marking analyzing thing, c) the isolated detection reagent through mark is hatched together with reagent composition, described reagent composition comprises: i) at least one coreagent, and ii) be selected from least one compound of the phosphoamide of formula I and formula II,
Formula I
R in formula I 1=CH 3, CH 2f, CH 2cl, CH 2cH 3, CHClCH 3, CH 2cH 2cl, C (CH 3) 2cH 3, CH 2cH 2cH 3, CClHCH 2cH 3or CH 2cH 2cH 2cH 3, R 2=H, and R 3=H,
Formula II,
D) galvanochemistry triggers luminous release, and e) measures electrochemiluminescence (ECL) signal, analyte thus.
An aspect of of the present present invention relates to the ECL method of the improvement based on reagent composition of the present invention, specifically, is characterized as the ECL method of low detectability.Described reagent composition unexpectedly strengthens specific signals and reduces background signal.More specifically, the inventive method is by being reduced in the sensitivity not having the background electrochemiluminescence in ECL label situation to be provided in the improvement of low detection level.
Present inventor unexpectedly finds to use some compound from phosphoamide to provide dramatic benefit, be included in the signal improved in ECL detection method and produce, and then the ECL improved measures performance.
A feature of the present invention is the method using electrochemiluminescence label to determine the analysis thing in sample to be studied, wherein adopts the one for measuring in the following method of electrochemiluminescence phenomenon.
Unexpectedly, uses the method being selected from the compound of phosphoamide to launch less background luminescence than the conventionally test reagent not containing these compounds.This is especially favourable when low detection level, wherein improves signal and substantially improves sensitivity with the ratio (=signal to noise ratio (S/N ratio)) of background.Unexpectedly, present inventor finds that using the inventive method to carry out electrochemiluminescence detects the signal noise ratio improve 10% to 60% causing ECL to detect.
The method detecting the analysis thing measured in sample by electrochemiluminescence according to the present invention can be carried out in one embodiment in aqueous.
In one embodiment, the phosphoamide used in the methods of the invention is selected from acetamide, 2-Fluorakil 100,2-chloroacetamide, propionamide, 2-chlorine propionamide, 3-chlorine propionamide, butyramide and 2-chlorobutamide.
In a preferred embodiment, the phosphoamide used in the methods of the invention is selected from acetamide, 2-chloroacetamide, propionamide and butyramide.
In a preferred embodiment, the phosphoamide used in the methods of the invention is selected from acetamide, propionamide and butyramide.
In a preferred embodiment, the concentration of the phosphoamide used in the methods of the invention is 0.01M to 0.25M.In a kind of further preferred embodiment, the concentration of the phosphoamide of use is 0.01M to 0.2M.In a kind of further preferred embodiment, the concentration of the phosphoamide of use is 0.01M to 0.1M.
In one embodiment, the inventive method is particularly suitable for detecting biomolecule in interested sample, as protein, polypeptide, peptide, fragments of peptides, hormone, peptide hormone, vitamin, provitamin, vitamin metabolism thing and amino acid.
Sample for the inventive method is fluid sample in one embodiment, such as, and whole blood, serum or blood plasma.Sample, or more specifically interested sample, in one embodiment, can comprise any body fluid and ight soil.In one embodiment, sample will be fluid sample, as saliva, stool extract, urine, whole blood, blood plasma or serum.In one embodiment, sample will be whole blood, blood plasma or serum.
The detection reagent that sample and electricity consumption chemiluminescent groups mark " a) is hatched " and can be carried out in same place with " b) make to be combined with the detection reagent through marking of analyzing thing and be not separated containing the detection reagent through marking analyzing thing " by step together that one skilled in the art will recognize that in the methods of the invention, such as, carry out in identical reactor.Described step (a) and (b) can be carried out with the automation process controlled by control device.
Non-specific sample component and containing analyze thing through mark detection reagent can remove with separation method in the step (b) in the inventive method.What be such as combined with analysis thing can use washing step to be separated with the detection reagent through mark not containing analysis thing.
Support that other fractions tested that the electrochemiluminescence analyzing thing detects also can be used in the inventive method.
An aspect of of the present present invention covers effective corrosion-resistant demand, such as, for longer-term storage reagent mixture and reagent composition.Suitable antiseptic reply ECL signal produces does not have impact or produces ECL signal in desirable situation have active influence.
As suitable preservative compounds, boric acid and/or borate are accredited as and effectively control bacterium and conk and unexpectedly enhance specificity ECL signal.Use and comprise boric acid and/or borate, as the ECL detection method of the reagent composition of antiseptic, there is positive effect beyond expectation, namely strengthen the specificity ECL signal produced.
In one embodiment, the present invention relates to the method being detected the analysis thing measured in sample by electrochemiluminescence, comprise the following steps: hatch together with detection reagent a) sample and electricity consumption chemiluminescent groups marked, b) make to be combined with analyze thing through mark detection reagent be not separated containing the detection reagent through marking analyzing thing, c) the isolated detection reagent through mark is hatched together with reagent composition, described reagent composition comprises: i) at least one coreagent, and ii) be selected from boric acid and boratory antiseptic, d) galvanochemistry triggers luminous release, and e) measure electrochemiluminescence (ECL) signal, analyte thus.
In one embodiment, the feature being detected the method for the analysis thing measured in sample by electrochemiluminescence is that the reagent composition produced for ECL signal comprises and is selected from boric acid and boratory antiseptic, the concentration of described antiseptic is 0.1% to 5%, preferred concentration is 0.5% to 4%, and more preferably concentration is 0.5% to 2%.
In one embodiment, the feature being detected the method for the analysis thing measured in sample by electrochemiluminescence is that the reagent composition produced for ECL signal comprises boric acid as antiseptic, the concentration of described antiseptic is 0.1% to 5%, preferred concentration is 0.5% to 4%, and more preferably concentration is 0.5% to 2%.
In one embodiment, the feature being detected the method for the analysis thing measured in sample by electrochemiluminescence is that the reagent composition produced for ECL signal comprises borate as antiseptic, the concentration of described antiseptic is 0.1% to 5%, preferred concentration is 0.5% to 4%, and more preferably concentration is 0.5% to 2%.
The further improvement of the signal to noise ratio (S/N ratio) during the method detecting the analysis thing measured in sample by electrochemiluminescence that present inventor unexpectedly finds to merge phosphoamide and boric acid and/or boratory effect in a reagent composition can cause ECL to detect.During phosphoamide and boric acid and/or the cumulative function of borate in a reagent composition cause ECL to detect, signal produces and improves at least 10%, 25% or 50%.
In one embodiment, the present invention relates to the method being detected the analysis thing measured in sample by electrochemiluminescence, comprise the following steps: hatch together with detection reagent a) sample and electricity consumption chemiluminescent groups marked, b) make to be combined with analyze thing through mark detection reagent be not separated containing the detection reagent through marking analyzing thing, c) the isolated detection reagent through mark is hatched together with reagent composition, described reagent composition comprises: i) at least one coreagent, ii) at least one compound of the phosphoamide of formula I and formula II is selected from, and iii) be selected from boric acid and boratory at least one antiseptic, d) galvanochemistry triggers luminous release, and e) measure electrochemiluminescence (ECL) signal, analyte thus.
In one embodiment, the feature being detected the method for the analysis thing measured in sample by electrochemiluminescence is that described reagent composition also comprises washing agent and buffering agent.
In one embodiment, the feature being detected the method for the analysis thing measured in sample by electrochemiluminescence is that described reagent composition also comprises salt and/or defoamer.
In one embodiment, the present invention relates to and carry out electrochemiluminescence method for measuring, wherein under the existence of reagent composition of the present invention, cause electrochemiluminescence.
Typical ECL measuring process for EDL immunoassays is included in multiple exchanges that ECL measures liquid and/or potpourri in cell (such as flow cell).Typical ECL measuring process is made up of several steps of explained later.
Those of skill in the art will appreciate that ECL measures cell and must nurse one's health (conditioned) or regeneration by measuring cell with reagent composition drip washing ECL of the present invention and additionally applying electromotive force before carrying out ECL detecting step.This step is the part using ECL to determine the process analyzing thing.Describe in EP1051621, measure at ECL in the process of analyte in cell during this conditioning step, the surface of the potential electrode of supporting signal generation forms one deck.
For typical ECL measuring process, enter ECL by fluid inlet channel and measure in lumen, reagent mixture introduced clean and measure in cell through the ECL of conditioning.This potpourri is sample, reagent and magnetic-particle hatch thing.Introduce the described potpourri measured in cell to be surrounded by reagent composition of the present invention, described reagent composition of the present invention flowed before and after described potpourri.
In such ECL immunoassays, comprise with electrochemiluminescence group mark and it is characterized in that the detection reagent of the compound-molecule for analyzing is combined with these magnetic-particles by a pair specific biochemical binding partners (such as Streptavidin and biotin).Magnetic-particle such as dressing has Streptavidin-polymkeric substance, and biotin is combined with compound-molecule.
Measure in cell at ECL, magnetic-particle is captured on the surface of electrode together with the compound-molecule of its mark combined in the magnetic field of magnet being arranged in described ate electrode.During the continuous flow of potpourri, apply magnetic field, hatch thing and/or reagent composition thus and to be measured lumen from ECL by fluid outlet channels and discharge.
After trapping magnetic particle, the reagent composition of the present invention containing ECL coreagent is introduced ECL in the next step and is measured in cell, thus with described reagent composition washing magnetic-particle.The removing of this washing step come to hatch described in self-electrode thing in conjunction with component, it disturbs electrochemical reaction potentially.
By applying electromotive force, galvanochemistry causes in the then release of electrochemiluminescence (ECL) signal, luminous intensity is by light sensors thus, and the measuring of concentration that can be used as the compound-molecule through mark be on the magnetic-particle of electrode surface is evaluated, thus this concentration measuring again as analyte concentration in sample.
After electrochemiluminescence detects, ECL measures cell and usually uses clean fluid drip washing.
Mentioned by the equipment carrying out detection method by electrochemiluminescence has in embodiment chapters and sections (embodiment 1,2 or 3), or be described in EP1892524 (A1).And described equipment can comprise the device of the temperature for control survey unit and/or liquid container.Measuring unit is interpreted as the unit wherein measuring electrochemiluminescence.Liquid container can be tank, also can be feed arrangement; Such as, the pipe of reagent solution is included in measuring unit during measuring.
composition:
An aspect of of the present present invention relates to the reagent composition of the improvement produced for ECL-signal, and it causes the signal to noise ratio (S/N ratio) improved.More specifically, by being reduced in background electrochemiluminescence when not having ECL label, reagent composition of the present invention provides the sensitivity when low detection level of improvement.Unexpectedly, inclusion compound such as the reagent composition of phosphoamide launches less background luminescence than the conventionally test reagent not containing these compounds.This is especially favourable when low detection level, wherein improves signal and substantially improves sensitivity with the ratio (=signal to noise ratio (S/N ratio)) of background.The reagent composition of this improvement comprises other compound from the compound of the group of phosphoamide and the method for support generation ECL.Unexpectedly, present inventor finds that using reagent composition of the present invention to carry out electrochemiluminescence detects the signal noise ratio improve 10% to 60% causing ECL to detect.
An aspect of of the present present invention relates to the reagent composition providing the ratio of high signal background in electrochemiluminescence measures.Signal difference between specific signals and background signal is improved.The advantageous combination (specifically, by using the compound being selected from phosphoamide) of ECL coreagent, pH buffering agent, washing agent and pH that the character of described improvement has been tested and appraised out is accomplished.
Described reagent composition provides and is suitable for effectively causing ECL label to launch ECL and measuring the environment of ECL label by measuring ECL delicately.Reagent composition of the present invention optionally comprises other component, other component described comprise antiseptic, washing agent, defoamer, ECL active matter, salt, for pH control acidity and alkali compounds (buffering agent), metallic ion and/or metal-chelator.Reagent composition of the present invention also can comprise the composition (it can mark with ECL label in some cases) of biologicall test, comprises binding reagents, enzyme, zymolyte, accessory factor and/or enzyme inhibitor.The present invention also comprises the mensuration reagent, composition, kit, system and the system components that comprise reagent composition of the present invention, and optional other measures component.The present invention also comprises use reagent composition of the present invention and carries out ECL method for measuring.
In one embodiment, the present invention relates to the reagent composition for determining ECL, it comprises the compound of phosphoamide i) being selected from formula I and formula II, and ii) at least one coreagent.
In one embodiment, the phosphoamide in reagent composition is selected from acetamide, 2-Fluorakil 100,2-chloroacetamide, propionamide, 2-chlorine propionamide, 3-chlorine propionamide, butyramide and 2-chlorobutamide.
In a preferred embodiment, described phosphoamide is selected from acetamide, 2-chloroacetamide, propionamide and butyramide.In further embodiment, described phosphoamide is selected from acetamide, propionamide and butyramide.Phosphoamide has the independent concentration being most appropriate to ECL humidification.As in experiment shown in (specifically, table 2,3 and 4), those of skill in the art know and select suitable concentration for the phosphoamide selected by reagent composition.Those of skill in the art know the method for the phosphoamide determination optimum concentration in reagent composition.
In one embodiment, the concentration of phosphoamide that described reagent composition comprises is 0.01M to 0.25M.In further embodiment, the concentration of the phosphoamide that described reagent composition comprises is 0.01M to 0.2M.In further embodiment, the concentration of the phosphoamide that described reagent composition comprises is 0.01M to 0.1M.
The coreagent of reagent composition is selected from tertiary amine (such as tripropylamine (TPA)), oxalates and persulfate in one embodiment.In a preferred embodiment, described coreagent is TPA.
Comprising the antiseptic stoping growth of microorganism when storing reagent composition may be favourable.In addition, suitable antiseptic is identified to control bacterium and conk thus can longer-term storage and use described reagent composition.Reagent composition of the present invention can comprise one or more antiseptics in addition.In one embodiment of the present invention, reagent composition comprises antiseptic (anticorrosion reagent).
In one embodiment, the present invention relates to the reagent composition for determining ECL, it comprises the compound of phosphoamide i) being selected from formula I and formula II, ii) at least one coreagent, and iii) at least one antiseptic.
Preferably, antiseptic produces ECL signal does not affect or has active influence.Art technology personnel Yi Zhi oxazolidine (such as, Oxaban A or 4,4-bis-Jia Ji oxazolidine), azide are compatible with ECL with relevant antiseptic.Usually 0.01% to 1% oxazolidine concentration is used in test agent.In one embodiment, described reagent composition comprises the antiseptic of Xuan Zi oxazolidine, preferred Oxaban A.In one embodiment, the concentration of the antiseptic that described reagent composition comprises is 0.01% to 1%, and described in another embodiment, the concentration of the antiseptic that reagent composition comprises is 0.1% to 1%.Use the potpourri of two or more antiseptics also may be favourable.
Phosphoamide 2-chloroacetamide (CAA) already mentioned in the text also has antiseptic function except its ECL signal humidification.
As mentioned above, an aspect of of the present present invention contains the demand adding and ECL signal is produced to the effective antiseptic not affecting or have active influence.As suitable mineral compound, boric acid and/or borate are accredited as and effectively control bacterium and conk.Unexpectedly, present inventor finds for determining that the boric acid that exists in the reagent composition of ECL and/or borate to produce ECL signal and do not have negative influence.Unexpectedly find to comprise boric acid or borate and to ECL signal production process, there is positive role as the reagent composition of antiseptic, namely improve specific signals.In addition, they high activity and be favourable to the problem of endanger safety thing or the relevant low degree of environmental concern.Contrary with other conventional antiseptic of some in reagent composition, boric acid or borate are not halogen-containing and do not discharge formaldehyde.The result of the boric acid existed in ECL signal produces is shown in embodiment chapters and sections such as embodiment 2.
In one embodiment, the present invention relates to the reagent composition for determining ECL, it comprises i) at least one coreagent, and iii) be selected from boric acid and boratory at least one antiseptic.
In one embodiment, the present invention relates to the reagent composition for determining ECL, it comprises i) at least one coreagent, and iii) antiseptic boric acid.
In one embodiment, the present invention relates to the reagent composition for determining ECL, it comprises i) at least one coreagent, and iii) antiseptic borate.
In one embodiment, the present invention relates to the reagent composition for determining ECL, it comprises the compound of phosphoamide i) being selected from formula I and formula II, ii) at least one coreagent, iii) be selected from boric acid and boratory at least one antiseptic.
In one embodiment, reagent composition of the present invention comprises boric acid or borate as antiseptic, and concentration is 0.1% to 5%, and preferred concentration is 0.5% to 4%, and especially preferred concentration is 0.5% to 2%.
In a preferred embodiment, reagent composition of the present invention comprises boric acid as antiseptic, and concentration is 0.1% to 5%, and preferred concentration is 0.5% to 4%, and especially preferred concentration is 0.5% to 2%.
In a preferred embodiment, reagent composition of the present invention comprises borate as antiseptic, and concentration is 0.1% to 5%, and preferred concentration is 0.5% to 4%, and especially preferred concentration is 0.5% to 2%.
Reagent composition of the present invention optionally also comprises other fractions tested.Other fractions tested is selected from least one washing agent, at least one signal strengthens compound, comprise the buffering agent of acidity and the alkaline reagent controlled for pH and water.
In one embodiment, the present invention relates to the reagent composition for determining ECL, it comprises the compound of phosphoamide i) being selected from formula I and formula II, ii) at least one coreagent, iii) at least one antiseptic, iv) buffering agent, v) at least one washing agent, vi) salt and/or defoamer and vii) optional other fractions tested.
The washing agent being applicable to reagent composition of the present invention is selected from following those: fatty acid alcohol ethoxylates, comprises PEG ether, such as polidocanol or other there is formula C xeO ythe PEG ether of (X=8 – 18 and Y=2-9), (isotridecyl gathers ((glycol ether) to Genapol n), (APG), OG (octyl group-β-D-glucopyranoside) and zwitterionic detergent are as Zwittergent3-12 or their potpourri.Washing agent uses with the concentration of 0.01% to 2%.Easily can determine the optium concentration of each washing agent.Optimum concentration be 0.05% to 1% those.
In one embodiment, reagent composition of the present invention comprises washing agent, and it is selected from polidocanol or other has formula C xeO ythe PEG ether of (X=8 – 18 and Y=2-9), OG (octyl group-β-D-glucopyranoside) or zwitterionic detergent are as Zwittergent3-12 or their potpourri.In a preferred embodiment, described reagent composition comprises washing agent, and it is selected from polidocanol, OG (octyl group-β-D-glucopyranoside) and Zwittergent3-12, or their potpourri.
Further, electrochemical luminescence signals also improves by adjustment pH to 6.0 ~ 8.0, preferably 6.0 ~ 7.5, especially preferably 6.2 ~ 6.9.This carries out easily by using the pH buffering agent being suitable for this scope well known by persons skilled in the art.In one embodiment, the buffering agent being suitable for described reagent composition comprises KOH and phosphoric acid (H 3pO 4).
Further, signal is by adding salt to strengthen, and described salt comprises inorganic salt as NaBr, NaCl, NaJ.Salt (specifically NaCl) adds by concentration 1mM ~ 1M, preferred 10mM ~ 100mM, most preferably 10mM ~ 50mM.
Avoid generation bubble or foam may be favourable, especially in HTS application.Therefore, may expect to add defoamer in reagent composition.Multiple business defoamer (comprising Antifoam o-30, Antifoam204, Antifoam A, Antifoam SE-15, Antifoam SO-25 and Antifoam289) can add in reagent composition of the present invention.
Reagent composition of the present invention can comprise ECL label.Described ECL label can be conventional ECL label.The example of ECL label comprises three-dipyridine-ruthenium (RuBpy) and other organometallics, and wherein metal is such as the metal from race VII and VIII, comprises Re, Ru, Ir and Os.Those skilled in the art use these ECL labels to carry out labelled analyte specific reagent with electricity consumption chemiluminescent groups, or electricity consumption chemiluminescent groups labelled analyte itself.In one embodiment, reagent composition of the present invention comprises through the analysis thing of mark and/or the analysis thing specific reagent through mark, wherein ECL label is selected from US5, the ECL label disclosed respectively in 310,687 (A) (BPRu=Ru (bpy) 2-bpyCO-OSu) US2003/0124572 (A1) (Sulfo-BPRu NHS ester), EP720614 (A1) (Bpy2-Ru-bpy-CO-UEEK-korks.-OSu) and WO2002/027317 (A2) (BPRu-(UE)-25-K and BPRu2-SK4).
Reagent used in reagent composition and composition thereof can provide with liquid, freezing, cryogenic refrigeration, evaporative freezing, freeze-drying, gas, solid or dried forms before use.At least before use reagent composition, reagent is dissolved in solvent.Reagent composition of the present invention will be aqueous solution.In a preferred embodiment, described reagent is soluble in water.
These preparations improved are especially valuable in high sensitivity measuring.In some embodiments of the present invention, the performance that ECL measures obtains even further improving by the best of breed that reagent composition and electrode form.Described suitable ECL electrode composition comprises the electrode of Ir, Pt or carbon.
These favourable combinations comprise the phosphoamide of aforementioned enhancing ECL and are selected from boric acid and boratory Suitable preservatives, and the two all has improves character.These comprise use the present invention disclose reagent composition time higher dynamic range and improvement from the ECL signal of binding label and the ratio of ECL background signal.The sensitivity of this raising is important, and such as in the mensuration benefiting from lower detectability, (such as TroponinT measures (TNThs; Order-No.:05092744), hepatitis-B envelope antigen measures (HBeAg; Order-No.:11820583), anti-thyrotropin receptor measures (anti-TSHR; Order-No.:04388780)-specifically see embodiment chapters and sections).
These reagent composition preparations improved can provide better precision, and this can cause lower detectability in ECL measures.
Another aspect of the present invention relates to and reducing costs, and this is the volume owing to decreasing needed for sample, test-specific reagent and/or test agent.The loss of signal of lower reagent volume is made up by using favourable reagent composition of the present invention.
Another aspect of the present invention relates to the system of the improvement comprising reagent composition of the present invention and equipment and/or is suitable for system and the equipment of the improvement carrying out the inventive method.
ECL signal produces and also can improve when combinationally using separately or mutually above-mentioned discovery.
reagent mixture:
For determining ECL, reagent composition of the present invention can be mixed to form reagent mixture with other compound.In one embodiment, the present invention relates to the reagent mixture for determining ECL, it comprises the reagent composition for determining ECL, described reagent composition comprises the compound of phosphoamide i) being selected from formula I and formula II, ii) at least one coreagent, iii) sample to be studied and iv) the detection reagent of at least one electricity consumption chemiluminescent groups mark.
In one embodiment, the present invention relates to the reagent mixture for determining ECL, it comprises the reagent composition for determining ECL, described reagent composition comprises the compound of phosphoamide i) being selected from formula I and formula II, ii) at least one coreagent, iii) at least one antiseptic, iv) the detection reagent of sample to be studied and v) at least one electricity consumption chemiluminescent groups mark.
In one embodiment, the present invention relates to the reagent mixture for determining ECL, it comprises the reagent composition for determining ECL, described reagent composition comprises the compound of phosphoamide i) being selected from formula I and formula II, ii) at least one coreagent, iii) be selected from boric acid and boratory antiseptic, iv) sample to be studied and v) at least one electricity consumption chemiluminescent groups mark detection reagent.
In further embodiment, the present invention relates to the reagent mixture for determining ECL, it comprises the reagent composition for determining ECL, described reagent composition comprises and i) is selected from boric acid and boratory antiseptic, ii) at least one coreagent, iii) sample to be studied and iv) the detection reagent of at least one electricity consumption chemiluminescent groups mark.In a preferred embodiment, described reagent mixture comprises antiseptic boric acid.In a preferred embodiment, described reagent mixture comprises antiseptic borate.
The present invention also relates to the reagent mixture for determining ECL in one embodiment, it comprises the reagent composition for determining ECL, described reagent composition comprises and i) is selected from boric acid and boratory antiseptic, ii) at least one coreagent, iii) sample to be studied, iv) washing agent, v) buffering agent, vi) the detection reagent of at least one electricity consumption chemiluminescent groups mark, and vii) comprise salt and/or defoamer.
In a kind of further preferred embodiment, the present invention relates to the reagent mixture for determining ECL, it comprises the reagent composition for determining ECL, described reagent composition comprises the compound of phosphoamide i) being selected from formula I and formula II, ii) at least one coreagent, iii) be selected from boric acid and boratory antiseptic, iv) sample to be studied, v) washing agent, vi) buffering agent and vii) the detection reagent of at least one electricity consumption chemiluminescent groups mark.
Reagent mixture also can comprise at least one washing agent and the buffering agent for control pH.Optionally, reagent mixture can comprise salt and/or defoamer.
Other fractions tested in reagent mixture is selected from unlabelled analysis thing specific reagent, analysis thing homologue, solid phase dressing and reduces the material of interference.
purposes:
An aspect of of the present present invention relate to of the present invention improve reagent composition and/or improve reagent mixture carrying out the purposes in electrochemical luminous detection method.
In one embodiment, the present invention relates to the phosphoamide being selected from formula I and formula II and carry out the purposes in electrochemiluminescence detection.In one embodiment, the present invention relates to the phosphoamide being selected from formula I and formula II and carry out the purposes in electrochemical luminous detection method operation.
In a preferred embodiment, the present invention relates to the phosphoamide being selected from acetamide, 2-Fluorakil 100,2-chloroacetamide, propionamide, 2-chlorine propionamide, 3-chlorine propionamide, butyramide and 2-chlorobutamide and carry out the purposes in electrochemiluminescence detection.
In the preferred embodiment of another kind, the present invention relates to the phosphoamide being selected from acetamide, 2-chloroacetamide, propionamide and butyramide and carrying out the purposes in electrochemiluminescence detection.In another embodiment, the present invention relates to the phosphoamide being selected from acetamide, propionamide and butyramide and carrying out the purposes in electrochemiluminescence detection.
In one embodiment, the present invention relates to reagent composition and determining the purposes in ECL, described reagent composition comprises the compound of phosphoamide i) being selected from formula I and formula II, ii) at least one coreagent, and iii) at least one antiseptic.
In one embodiment, the present invention relates to reagent composition and determine the purposes in ECL, described reagent composition comprises the compound of phosphoamide i) being selected from formula I and formula II, ii) at least one coreagent, and iii) be selected from boric acid and boratory antiseptic.
In one embodiment, the present invention relates to reagent composition and determine the purposes in ECL, described reagent composition comprises compound i) being selected from phosphoamide, described phosphoamide is selected from acetamide, 2-Fluorakil 100,2-chloroacetamide, propionamide, 2-chlorine propionamide, 3-chlorine propionamide, butyramide and 2-chlorobutamide, ii) at least one coreagent, and iii) antiseptic.
In one embodiment, reagent composition of the present invention is suitable for conditioning or regeneration ECL measurement cell and determines ECL signal.In one embodiment, described reagent composition is used as conditioning solution.In one embodiment, reagent composition of the present invention is used for conditioning or regeneration ECL measurement cell.In one embodiment, described reagent composition is used for conditioning or regeneration ECL measures cell, it comprises the compound being selected from phosphoamide, and described phosphoamide is selected from acetamide, 2-Fluorakil 100,2-chloroacetamide, propionamide, 2-chlorine propionamide, 3-chlorine propionamide, butyramide and 2-chlorobutamide.In another embodiment, described reagent composition is used for conditioning or regeneration ECL measures cell, and it comprises the compound being selected from phosphoamide, and described phosphoamide is selected from acetamide, propionamide and butyramide.
For for carrying out electrochemical luminous detection method, described reagent composition can be mixed to form reagent mixture with other compound, and described other compound such as sample to be studied, at least one have the detection reagent of electrochemiluminescence group and support other component following of the method.
In one embodiment, the present invention relates to the reagent mixture comprising reagent composition and determine the purposes in ECL, a) compound of phosphoamide i) being selected from formula I and formula II is comprised, ii) at least one coreagent, and iii) antiseptic, b) sample to be studied, and c) at least one electricity consumption chemiluminescent groups mark detection reagent.
The reagent mixture that the present invention relates to the reagent composition comprised for determining ECL is determining the purposes in ECL, a) compound of phosphoamide i) being selected from formula I and formula II is comprised, ii) at least one coreagent, and iii) be selected from boric acid and boratory antiseptic, b) sample to be studied, and c) at least one electricity consumption chemiluminescent groups mark detection reagent.
In further embodiment, the present invention relates to boric acid or borate is carrying out the purposes in electrochemiluminescence detection.Also in one embodiment, the present invention relates to and be selected from boric acid and boratory antiseptic is carrying out the purposes in electrochemical luminous detection method operation.
In one embodiment, the present invention relates to reagent mixture and determine the purposes in ECL, described reagent mixture comprises a) for determining the reagent composition of ECL, its comprise i) be selected from boric acid and boratory antiseptic and ii) at least one coreagent, b) sample to be studied, and c) at least one electricity consumption chemiluminescent groups mark detection reagent.
In addition, for determining that the reagent mixture of ECL can comprise the component being selected from washing agent and the buffering agent for pH control.Optionally, reagent mixture used can comprise salt and/or defoamer.Other fractions tested in reagent mixture is selected from unlabelled analysis thing specific reagent, analysis thing homologue, solid phase dressing and reduces the material of interference.
kit:
An aspect of of the present present invention relates to the kit of one or more components comprising reagent composition of the present invention in one or more containers.These components optionally merge with other reagent and form reagent composition of the present invention.Kit also can comprise mensuration reagent, thinning agent, wash solution, protein denaturation reagent, enzyme, binding reagents, assay plate, the disposables of other mensuration related component such as ECL label, ECL mark in one embodiment, etc.
In one embodiment, described reagent composition is contained in one or more glass or plastic containers, suitably indicates about the information of reagent composition content with about the correct explanation storing and use.About in the RFID chip that the information of reagent composition content, lot number, date of manufacture, shelf-life and also can being stored in about the correct explanation stored and use is placed in glass or plastic containers.The information that described RFID chip stores reads by the antenna be connected with RFID reader and processes further in control device.
In one embodiment, some or all components of described reagent composition can store with liquid or drying regime in one embodiment.
In one embodiment, the present invention relates to the kit measuring ECL, it comprises the reagent composition for determining ECL, and described reagent composition comprises compound and the ii of phosphoamide i) being selected from formula I and formula II) at least one coreagent.
In a preferred embodiment, the present invention relates to the kit measuring ECL, it comprises the reagent composition for determining ECL, and described reagent composition comprises the phosphoamide and ii that i) are selected from acetamide, 2-Fluorakil 100,2-chloroacetamide, propionamide, 2-chlorine propionamide, 3-chlorine propionamide, butyramide and 2-chlorobutamide) at least one coreagent.
In the preferred embodiment of another kind, the present invention relates to the kit measuring ECL, it comprises the reagent composition for determining ECL, and described reagent composition comprises the phosphoamide and ii that i) are selected from acetamide, 2-chloroacetamide, propionamide and butyramide) at least one coreagent.
In one embodiment, the present invention relates to the kit measuring ECL, it comprises the reagent composition for determining ECL, and described reagent composition comprises the compound of phosphoamide i) being selected from formula I and formula II, ii) at least one coreagent and iii) antiseptic.
In a preferred embodiment, the present invention relates to the kit measuring ECL, it comprises the reagent composition for determining ECL, described reagent composition comprises the compound of phosphoamide i) being selected from formula I and formula II, ii) at least one coreagent and iii) be selected from boric acid and boratory antiseptic.
In the preferred embodiment of another kind, the present invention relates to the kit measuring ECL, it comprises the reagent composition for determining ECL, described reagent composition comprises phosphoamide i) being selected from acetamide, 2-Fluorakil 100,2-chloroacetamide, propionamide, 2-chlorine propionamide, 3-chlorine propionamide, butyramide and 2-chlorobutamide, ii) at least one coreagent and iii) be selected from boric acid and boratory antiseptic.
In the preferred embodiment of another kind, the present invention relates to the kit measuring ECL, it comprises the reagent composition for determining ECL, described reagent composition comprises phosphoamide i) being selected from acetamide, 2-chloroacetamide, propionamide and butyramide, ii) at least one coreagent and iii) be selected from boric acid and boratory antiseptic.
In one embodiment, the present invention relates to and measure the kit of ECL, it comprises the reagent composition for determining ECL, described reagent composition comprise at least i) be selected from boric acid and boratory antiseptic and ii) at least one coreagent.
Aforesaid measurement itself has significantly improved known operation.And, may improve by merging these measurements the sensitivity and/or range of dynamic measurement of analyzing thing detection assay further significantly.
There is provided following examples and figure to help understand the present invention, true scope of the present invention is listed in the dependent claims.Should be appreciated that and under the prerequisite not deviating from purport of the present invention, amendment can be made to listed operation.
embodiment 1
The ECL of the mensuration damping fluid (reagent composition) containing phosphoamide is used to measure
ECL measures and uses Roche 2010 equipment carry out, and wherein adopt the following code that can be used for this mensuration.
The compound being selected from phosphoamide of concentration as shown in table 2,3 and 4 is added in following mensuration damping fluid:
180mM tripropylamine (TPA)
0.1% polidocanol
300mM phosphate buffer
Final pH uses KOH/H 3pO 4be adjusted to pH6.8.Measure damping fluid and also can be used as blank value.
The compound (chemical formula of phosphoamide is shown in Table 1) being selected from phosphoamide is added in mensuration damping fluid (reagent composition) with prescribed concentration.Result is reported as relative to using the signal recovery lacking the measurement of the mensuration damping fluid of these compounds.
The mensuration Buffer background carrying out the mensuration damping fluid of the phosphoamide containing concentration shown in table 2 is measured.Value lower than 100% represents that adding selected phosphoamide with prescribed concentration reduces ECL background signal.Being especially favourable not having when reducing background electrochemiluminescence at low detection level in ECL label situation, wherein improving the sensitivity that signal and the ratio (=signal to noise ratio (S/N ratio)) of background significantly improve mensuration.
Table 2:
N.d.=does not determine
In similar experiment, determine the signal of free label.The solution that the representative of free label value comprises free ECL label is not having the signal that in fine-grained situation, (in mensuration damping fluid, 10nM RuBpy) produces.This value is listed in table 3 with percentage relative to the mensuration damping fluid without any other compound.This mensuration form is also referred to as homogeneous phase measurement or homogeneous determination form.Value higher than 100% represents that adding selected phosphoamide with selected concentration enhances ECL signal.Result is shown in Table 3.
Table 3:
N.d.=does not determine
In addition, the value determining to adopt the simplification comprising pearl to measure.This labor measurement is the mensuration for high specific signal of the particulate comprising RuBpy mark.This mensuration form is also referred to as heterogeneous measurement or heterogeneous assay formats.The difference between specificity labor measurement-signal and background signal (mensuration Buffer background) of use said determination damping fluid and other compound is expressed as the Δ labor measurement relative to the mensuration damping fluid without any other compound in %.Value higher than 100% represents that adding selected phosphoamide with selected concentration enhances ECL signal.Result is shown in Table 4.
Table 4:
N.d.=does not determine
Illustrate the result of the propionamide as shown in table 2,3 and 4 in FIG.Illustrate the result of the 2-chloroacetamide as shown in table 2,3 and 4 in fig. 2.Illustrate the result of the butyramide as shown in table 2,3 and 4 in figure 3.Illustrate the result of the acetamide as shown in table 2,3 and 4 in the diagram.
embodiment 2
Boric acid strengthens antiseptic as signal
ECL measures use 2010 equipment adopt following mensuration to recommend code to carry out.
Following ECL measures damping fluid and is used for determining blank value:
180mM tripropylamine (TPA)
0.1% polidocanol
0.1%Oxaban A
300mM phosphate buffer
The boric acid of increment as noted is added in this mensuration damping fluid.Use KOH/H 3pO 4final pH is adjusted to pH6.8.
Carry out mensuration Buffer background with the mensuration damping fluid of the boric acid containing concentration shown in table 5 to measure.The representative of free label value not to have in fine-grained situation (in mensuration damping fluid by the solution containing free ECL label, 10nM RuBpy, homogeneous phase measurement) signal that produces, its relative to the mensuration damping fluid without any other compound in %.This labor measurement is the mensuration of the particulate comprising RuBpy mark for high specific signal.In-vitro diagnosis as business measures, tSH measures (for thyrotropin assay; Order-No.:11731459) for determining Δ TSH.TSH graduator 1 (TSH Cal set; Order-No.:04738551) as low-level graduator (there is not analysis thing), in measuring for TSH, background signal (TSH Cal1) is provided; TSH graduator 2 provides high signal value (TSH Cal2) in measuring for TSH.
Labor measurement, TSH Cal1 and TSH Cal2 the results are depicted in Fig. 5, measure the relative recovery (in %) of damping fluid as the object of reference not adding boric acid.
Specifically, following measurement is carried out.
Table 5:
Add boric acid and improve the heterogeneous signal of specificity as antiseptic in mensuration damping fluid, in labor measurement and determine in TSH Cal2 especially true.
embodiment 3
Mensuration damping fluid pair containing propionamide and boric acid the impact of the lower detectability measured
Determine several business external test (HBeAg:Roche Order-No.:11820583, anti-TSHR:Roche Order-No.:04388780, TNThs:Roche Order-No.:05092744) lower detectability with compare two kinds measure buffer formulation.
Measure buffer A:
180mM TPA, 0.1% polidocanol, 300mM phosphate buffer, 0.1%Oxaban A
Measure buffer B:
180mM TPA, 0,1% polidocanol, 50mM propionamide, 300mM phosphate buffer, 1% boric acid
The final pH measuring buffer A and B uses KOH/H 3pO 4be adjusted to pH6.8.
Above-mentioned three kinds of commercially available mensuration are analyzed, to show phosphoamide and propionamide and antiseptic and boric acid to the impact of mensuration performance detecting low-down analyte concentration.
Be determined at analyser is measured and calibrates as described in its packing plug.In order to calculate lower detectability, determine the signal of the sample do not analyzed thing (HBeAg, anti-TSHR) or there is pole harmonic analysis substrate concentration (TNThs).Calculate the standard deviation that 21 samples (21-fold) are determined, be multiplied by 2 (2SD) or 3 (3SD), and be added (HBeAg, TNThs) with the average of signal or subtract each other (anti-tsh R, competition assay).Then the calibration curve of each mensuration is used to determine the corresponding concentration calculating signal.For the sample (TNThs) with harmonic analysis substrate concentration, the analyte concentration of sample deducts from these calculating concentrations.
These 3 kinds of mensuration are benefited from and are improved reagent composition containing phosphoamide and containing the of the present invention of boric acid (boric acid also has antiseptic function).The result that HBeAg, anti-TSHR and TNThs measure is shown in table 6,7 and 8.
Table 6:
Table 7:
Table 8:
Accompanying drawing explanation
Fig. 1: concentration is the measurement result of the propionamide (X-axle) of 0.001M to 0.25M; The relative recovery (percentage relative to object of reference) measuring Δ labor measurement (labor measurement-mensuration Buffer background), mensuration Buffer background and free label mensuration is shown in Y-axle.Specifically see embodiment 1.
Fig. 2: concentration is the measurement result of the 2-chloroacetamide (X-axle) of 0.001M to 1M; The relative recovery (percentage relative to object of reference) measuring Δ labor measurement (labor measurement-mensuration Buffer background), mensuration Buffer background and free label mensuration is shown in Y-axle.Specifically see embodiment 1.
Fig. 3: concentration is the measurement result of the butyramide (X-axle) of 0.001M to 1M; The relative recovery (percentage relative to object of reference) measuring Δ labor measurement (labor measurement-mensuration Buffer background), mensuration Buffer background and free label mensuration is shown in Y-axle.Specifically see embodiment 1.
Fig. 4: concentration is the measurement result of the acetamide (X-axle) of 0.001M to 1M; The relative recovery (percentage relative to object of reference) measuring Δ labor measurement (labor measurement-mensuration Buffer background), mensuration Buffer background and free label mensuration is shown in Y-axle.Specifically see embodiment 1.
Fig. 5: concentration is the measurement result of the boric acid (X-axle) of 0 to 5%; Labor measurement is used as the example of high specific signal; TSH graduator 1 provides background signal (TSH Cal1) as low-level graduator; TSH graduator 2 provides signal (TSH Cal2) in high detection level.Result is not to add the percentage mapping of the object of reference reagent composition of boric acid.Specifically see embodiment 2.

Claims (16)

1. detected the method for the analysis thing measured in sample by electrochemiluminescence, comprise the following steps:
A) hatch together with detection reagent sample and electricity consumption chemiluminescent groups marked,
B) make to be combined with analyze thing through mark detection reagent be not separated containing the detection reagent through marking analyzing thing,
C) hatched together with reagent composition by the isolated detection reagent through mark, described reagent composition comprises:
I) at least one coreagent, and
Ii) at least one compound of the phosphoamide of formula I and formula II is selected from,
In formula I, R 1=CH 3, CH 2f, CH 2cl, CH 2cH 3, CHClCH 3, CH 2cH 2cl, C (CH 3) 2cH 3, CH 2cH 2cH 3, CClHCH 2cH 3or CH 2cH 2cH 2cH 3, R 2=H, and R 3=H,
D) galvanochemistry triggers luminous release, and
E) electrochemiluminescence (ECL) signal is determined, analyte thus.
2. the method for claim 1, is characterized in that the analysis thing using ECL to measure in sample carries out in aqueous.
3. the method for claim 1 or 2, is characterized in that described phosphoamide is selected from acetamide, 2-Fluorakil 100,2-chloroacetamide, propionamide, 2-chlorine propionamide, 3-chlorine propionamide, butyramide and 2-chlorobutamide.
4. the method any one of claim 1 to 2, is characterized in that described reagent composition comprises the phosphoamide that concentration is 0.01M to 0.25M.
5. the method any one of claim 1 to 2, is characterized in that step c) reagent composition comprise antiseptic.
6. the method for claim 5, is characterized in that step c) reagent composition comprise the antiseptic that concentration is 0.1% to 5%.
7. the method for claim 1 or 2, is characterized in that step c) reagent composition comprise and be selected from boric acid and boratory antiseptic.
8. the method any one of claim 1 to 2, is characterized in that step c) reagent composition comprise washing agent and buffering agent.
9. the method for claim 8, is characterized in that step c) reagent composition also comprise salt and/or defoamer.
10., for determining the reagent composition of electrochemiluminescence, it comprises:
I) compound of the phosphoamide of formula I and formula II is selected from, and
Ii) at least one coreagent,
Wherein said coreagent is selected from tertiary amine, oxalates and persulfate,
The compound of the phosphoamide of described formula I is:
In formula I, R 1=CH 3, CH 2f, CH 2cl, CH 2cH 3, CHClCH 3, CH 2cH 2cl, C (CH 3) 2cH 3, CH 2cH 2cH 3, CClHCH 2cH 3or CH 2cH 2cH 2cH 3, R 2=H, and R 3=H,
The compound of the phosphoamide of described formula II is:
11. the reagent composition of claim 10, it is characterized in that described tertiary amine is tripropyl amine (TPA).
12. the reagent composition of claim 10, it is characterized in that described reagent composition also comprises and be selected from boric acid and boratory antiseptic.
13. for determining the reagent mixture of electrochemiluminescence, and it comprises the detection reagent of the reagent composition of claim 10 or 12, sample to be studied and at least one electricity consumption chemiluminescent groups mark.
14. the reagent composition of claim 10 or 12 is determining the purposes in electrochemiluminescence.
15. phosphoamides being selected from formula I and formula II are carrying out the purposes in electrochemiluminescence detection operation,
The compound of the phosphoamide of wherein said formula I is:
In formula I, R 1=CH 3, CH 2f, CH 2cl, CH 2cH 3, CHClCH 3, CH 2cH 2cl, C (CH 3) 2cH 3, CH 2cH 2cH 3, CClHCH 2cH 3or CH 2cH 2cH 2cH 3, R 2=H, and R 3=H,
The compound of the phosphoamide of described formula II is:
16. measure the kit of electrochemiluminescence, it comprises the reagent composition of claim 10 or 12.
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